Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has raised public concerns.The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses;thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed.Our previous studies demonstrated that the strategy by replacing H3 subtype hemagglutinin (H-A) transmembrane domain (TMD) with H1, H5 or H9 HA TMD could enhance H1, H5 or H9 HA based crossprotection.In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM, in which TMD was replaced with H3 HA TMD.We investigated whether the TMD replacement could increase H7 HA protein based cross-protection against different genotypes of H7N9 viruses.The results showed that the H7-53TM either with or without MF59 adjuvant induced increased HI antibodies to a panel of H7N9 viruses in mice.Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection.Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and moderate pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT.These results demonstrated that replacement of the TMD of H7 HA with H3 HA TMD presented an enhanced crossprotection, suggesting that this approach can be used for vaccination against divergent H7N9 viruses.